Site-specific mutagenesis is one of the most important experimental tools available for protein research, serving as the cornerstone for selective structural modification in both basic mechanistic enzymology and commercial genetic engineering. Despite its tremendous significance, this technique suffers from the limitation that amino acid substitutions are restricted to the twenty primary amino acids. This prerequisite normally excludes the direct site-specific introduction into proteins of "designer" amino acids intended to modify function or activity in a predictable way. This proposal requests funding to continue our work on developing and applying techniques for incorporation of nonnatural amino acids into proteins. In the previous four years we have assessed the generality of using semi- synthetic suppressors in conjunction with in vitro translation systems for this purpose. We have also begun several collaborative projects in which this technique is being used to prepare mutant proteins for structure/function studies of three different proteins. During the period of funding requested, we will carry out some fine-tuning of current procedures, including efforts to i) improve efficiency of suppressor synthesis and ii) develop general methods for increasing protein production and/or suppression efficiency. The primary focus will be on applying the technique to: iii) study the protein-protein binding interaction between FK506-FKBP complex and calcineurin via photochemical crosslinking, iv) investigate electron transfer from cytochrome C to cytochrome C peroxidase (CCP) mutants in which proposed hydrogen bond pathways have been disrupted by, in effect, single atom mutations, and v) investigate the introduction of intrinsic fluorescence probes at single sites, initially applied to preparing mutants of soluble tissue factor (sTF).